Over the past several decades, numerous basic and clinical efforts to treat various diseases including cancer have been made globally, and research on signal transduction based on the action mechanisms of genes has been actively conducted to develop new therapeutic agents.
Until now, while many natural substances, protein/peptide therapeutic agents and chemically-synthetic therapeutic agents have been developed and used, in many cases, the reoccurrence of diseases even after the treatment with such therapeutic agents may occur, there may have serious side effects also on normal cells in organisms, and such therapeutic agents may not properly work depending on patients even with the same type of disease due to a variety of the causes of a disease. In addition, there is resistance to a therapeutic agent or a recurrent disease appears after the treatment with a therapeutic agent.
To date, most therapeutic agents are based on the inhibition of signaling receptors present on the surface of cancer cells and kinases/dephosphorylases important in intracellular signal transduction. However, since such receptors and enzymes are involved in the survival of normal cells, the developed anticancer agents may affect normal cells and thus have serious side effects.
Recently, the development of therapeutic agents targeting transcription factors and epigenomes, which are involved in fundamental regulation of gene expression, is also growing. However, such factors are also needed to maintain normal cells in the organisms, and therefore it is difficult to find effective targets.
An Mi-2/NuRD chromatin remodeling complex is induced by DNA methylation, thus exhibits a function of maintaining the inhibition of transcription, and includes HDAC as a component. Since the Mi-2/NuRD complex may directly bind to a DNA methylase as well as a DNA methylation site, it plays a very important role in epigenetically inhibiting gene expression, and MBD2 knock-out (MBD2 −/−) mice are known to show normal survival and proliferation and not exhibit harmful effects (Hendrich B, Guy J, Ramsahoye B, Wilson V A, Bird A, Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes Dev, 2001, 15: 710), and when MBD2 expression is decreased in cancer cell line and cancer-implanted animal models, it is known to have an inhibitory effect on cancer growth (Slack A, Bovenzi V, Bigey P, Ivanov M A, Ramchandani S, Bhattacharya S, tenOever B, Lamrihi B, Scherman D, Szyf M, Antisense MBD2 gene therapy inhibits tumorigenesis. J Gene Med, 2002, 4: 381; Sansom O J, Berger J, Bishop S M, Hendrich B, Bird A, Clarke A R, Deficiency of Mbd2 suppresses intestinal tumorigenesis. Nat Genet, 2003, 34: 145; Mian O Y, Wang S Z, Zhu S Z, Gnanapragasam M N, Graham L, Bear H D, Ginder G D, Methyl-binding domain protein 2-dependent proliferation and survival of breast cancer cells. Mol Cancer Res, 2011, 9: 1152).
In previous research on a globin gene transcription regulatory mechanism by the transcription factor CP2c (also named TFCP2, LSF, LBP1, and USF) in murine erythroleukemia (MEL) cell line models, the inventors of the present invention confirmed that the attenuation of MBD2 expression is essential for normal erythrocyte differentiation. MEL cell lines are cancer cells in which differentiation is stopped in a proerythroblast stage during erythrocyte differentiation, but when the culture solution is treated with a chemical inducer such as dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), globin gene expression occurs as well as terminal differentiation.
They also confirmed that CP2c is involved in erythrocyte-specific globin gene transcription by forming a CBP complex with CP2b and PIAS1 proteins (Kang H C, Chae J H, Lee Y H, Park M A, Shin J H, Kim S H, Ye S K, Cho Y S, Fiering S, Kim C G, Erythroid cell-specific alpha-globin gene regulation by the CP2 transcription factor family Mol Cell Biol, 2005, 25: 6005; Kang H C, Chae J H, Jeon J, Kim W, Ha D H, Shin J H, Kim C G, Kim C G, PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific alpha-globin expression. Nucleic Acids Res, 2010, 38; 5456), and identified that p66α (GATAD2A), one of the components of Mi-2/NuRD CRC, directly binds to CP2c (Kang H C, Chung B M, Chae J H, Yang S I, Kim C G, Kim C G, Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2. FEBS J, 2005, 272: 1265).
In addition, p66α inhibits the transcription activity of CBP transcription factor complexes (CP2c, CP2b and PIAS1) through binding with CP2c, and when the p66α expression-attenuated MEL cell line is intravenously injected into immunodeficient mice, tumorigenesis in the blood, spleen and liver were remarkably inhibited as well as splenomegaly shown in normal control cells. It was confirmed that the p66α expression is constantly maintained during the induction of erythrocyte differentiation in the MEL cells and erythrocyte differentiation in the bone marrow, and the expression of MBD2 as another component of Mi-2/NuRD CRC, which is known to directly bind to p66α, is rapidly reduced.
Actually, the transcription activity of the CBP complex had an inverse-correlation with MBD2 expression, and the MBD2 expression-attenuated MEL cell line showed spontaneous erythrocyte differentiation. In addition, it was confirmed that MBD2 is involved in the activity of the CBP complex by the interaction with p66α, and a new fact that Mi-2/NuRD CRC present in undifferentiated MEL cells, as typical CRC having MBD2 (restrictive Mi-2/NuRD CRC), inhibits the expression of a target gene, but permissive Mi-2/NuRD CRC that does not have MBD2 aids the transcription activity of the CBP complex while not separated from a globin gene promoter during normal erythrocyte differentiation was identified.
As such, since MBD2 does not affect the survival of normal cells, and the MBD2-p66α interaction is important for the inhibitory function of Mi-2/NuRD CRC against gene expression, it is determined that MBD2 may be an important target for the development of an anticancer agent.